Baicalein is one of the major flavonoids obtained from the Scutellaria root. Previous pharmacological studies found that baicalein had neuroprotective effects in animal models of Parkinson's disease. The purpose of this paper was to explore the molecular mechanism of the action of baicalein on PC12 cells and isolated rat brain mitochondria. Firstly, we investigated the effects of baicalein on rotenone-induced toxicity in PC12 cells. The results showed that baicalein suppressed rotenone-induced apoptosis, and inhibited the accumulation of reactive oxidant species, ATP deficiency, mitochondrial membrane potential dissipation, and caspase-3/7 activation in a concentration-dependent manner, indicating that baicalein likely improved mitochondrial function. Furthermore, we used isolated rat brain mitochondria to evaluate the effect of baicalein. Treatment with baicalein prevented rotenone-induced reactive oxidant species production, ATP deficiency and mitochondrial swelling in isolated brain mitochondria. Interestingly, exposure of isolated mitochondria to baicalein promoted mitochondrial active respiration. These results suggest that baicalein may be a mitochondria-targeted antioxidant and exerts neuroprotective effects on rotenone-induced neurotoxicity. This study supports our previous research that baicalein possesses neuroprotective activity in vivo and it is worthy of further study.